A number of serious diseases affecting the human population can be closely associated with the improper aggregation of polypeptide fragments and characterized by aggregate deposition in lesions or plaques often resulting in abnormal physiological function at the plaque site. For instance, a stretch of polyglutamine repeats in a particular protein has been proposed as the cause for many neurological disorders including Huntington's disease and spinal bulbar muscular atrophy, while aggregation of other unrelated proteins are cited as the causes of prion disease (PrP aggregation), Parkinson's disease and amyotrophic lateral sclerosis (ALS) (α-synuclein aggregation), dialysis-related amyloidosis (β-2 microglobulin aggregation), corneal dystrophy (kerato-epithelial deposits), and aggregation of islet amyloid polypeptide in more than 95% of type II diabetes. Of particular interest is the aggregation of β-amyloid polypeptides in Alzheimer's Disease.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the gradual decline of cognitive function and memory loss associated with amyloid plaque formation, neurofibrillary tangles, and associated neuronal toxicity. The initiation and onset of Alzheimer's disease is believed to have both genetic origins as well as sporadic, environmentally influenced onset (Davies P., Annals of the N.Y. Acad Of Sci. 924: 8-16.2000). Several genetic mutations have been characterized to date that are associated with familial onset AD. Mutations which are significantly involved in AD onset have been found in the Amyloid Precursor Protein (APP) itself, and in either the presenilin-1 or presenilin-2 genes. However, the common denominator in all of these mutations is the formation of protein, or senile plaques derived from a cleavage fragment, designated amyloid beta (Aβ), of the APP molecule which is deposited in the brain of affected individuals, and results in toxicity and death of neuronal cells. This phenomenon of plaque formation can also be detected in another genetic defect not directly related to AD, the trisomy of chromosome 21 involved in Down's syndrome.
Amyloid precusor protein (APP), a single transmembrane glycoprotein possessing a large cytoplasmic domain and a short intracellular C-terminal region, occurs naturally as several splice forms of either 751 amino acids (Ponte et al., Nature 331: 525-527. 1988; Tanzi et al., Nature 331: 528-530. 1988), 770 amino acids (Kitaguchi et. al., Nature 331: 530-532. 1988), or 695 amino acids (Kang et. al., Nature 325: 733-736. 1987), designated as “normal” APP. The APP695 variant is more widely expressed in neurons.
Amyloid precursor protein cleavage takes place through a series of enzymatic reactions mediated by the α, β, and γ secretases. The α-secretase cleaves ˜12 residues N-terminal of the transmembrane domain of APP, at approximately residue 687 of APP770, generating the large soluble protein s-APPα, which is nonpathogenic, and a C-terminal fragment of 83 amino acids. The γ-secretase cleaves after amino acid 711 or 712 in the C-terminal end to create a free peptide termed p3. Alternatively, APP can be cleaved N-terminal to the transmembrane domain before aspartyl residue 672 by β-secretase enzyme, a member of the aspartyl protease family of enzymes (Yan et al., Nature 402: 533-537. 1999), forming a truncated verison of sAPPα, referred to as sAPPβ. The remaining C-terminal fragment can also be cleaved by a γ-secretase, near residue 711, giving rise to soluble Aβ peptide. The γ-secretase generates an Aβ peptide of either 1-39, 1-40, 1-42, or 1-43 amino acids (where position 1 immediately follows the β-secretase cleavage site) depending upon its cleavage site. Aβ1-40 is the more abundant cleavage product produced by most cell types.
In addition to the formation of senile plaques by Aβ fragments, Aβ deposition can also be detected in nonfibrillar, granular associations termed diffuse plaques (Tagliavini et al., Neurosci. Lett. 93: 191). Diffuse Aβ plaques are detectable in brains of normal, healthy individuals, while very few senile, amydyloidogenic plaques are detected in the brains of non-AD affected individuals. Antibody staining against Aβ peptide revealed that the diffuse plaques are composed primarily of the highly amyloidogenic Aβ1-42 (Iwatsubo et al., Neuron 13:45. 1994) while senile plaques contain a mixture of both Aβ1-42 and Aβ1-40 peptides.
Several mutations of amyloid precursor protein that result in increased cleavage of APP into Aβ peptides have been characterized. The “Swedish” mutation, at amino acid residues KM 670/671→NL (of APP770), enhances the production of both Aβ1-40 and Aβ1-42 (Citron et al., Nature 360:6724. 1992; Lannfelt et al., Neurosci Lett 153: 85-7. 1993). The “London” mutation at residue 717, V→I, G, or F, also results in increased production of Aβ peptide fragments (Schenk et al., Nature 400: 173-177. 1999). Several other mutations have been identified, all of which cluster around one of the three secretase cleavage sites in APP, all leading to increased Aβ cleavage.
Many of the therapies contemplated for the treatment of AD target the formation of Aβ peptides by secretase enzyme activity, particularly β and γ secretases involved in the cleavage of APP into Aβ peptides. Cleavage of APP into Aβ is a natural enzymatic reaction that generates Aβ peptides in areas not associated with the neuronal damage such as basement membrane and arterioles and venules, and areas of the brain not associated with AD pathology. These deposits of Aβ are generally diffuse in nature rather than fibrillary, and Aβ is constitutively secreted by cells throughout life and is found in the cerebrospinal fluid and plasma of all normal individuals (Haas et al., Nature 359: 322-5. 1992; Seubert et al., Nature 359: 325-7. 1992). These and other data suggest that Aβ aggregation (as opposed to Aβ formation) represents another target for therapeutic intervention.
One recent approach for therapeutic intervention into Alzheimer's Disease and other diseases associated with polypeptide aggregation is treatment with agents that inhibit the nucleation/aggregation of polypeptides. Several screening assays are currently available for the detection of aggregating proteins or aggregating polypeptides involved in various debilitating human diseases.
A unique method for detecting aggregation of proteins is termed Time Resolved Anisotrpy Measurements (TRAMS) (Allsop et al, Biochem. Biophy. Res. Comm. 285: 58-63), which measures the movement of fluorescent particles in solution. TRAMS require a mixture of a fluorescently-labeled peptide and non-labeled peptide which are then mixed to the desired concentration, and anisotropy measurements taken over a course of time points. For this particular assay a modified single photon counter with a light emitting diode with a repetition rate of 1 MHz is used for measuring the light emission spectra. Slower movement of the fluorescing particles over time correlates with an increasing number of aggregates. While the TRAMS assay may measure the initial steps in protein aggregation, it is a very complex method of detecting peptide complexes which requires equipment not readily available and involves difficult interpretation of the data.
A scintillation proximity assay (SPA) can also be used to assess aggregation of β-amyloid polypeptides. In the SPA method, three species of β-amyloid1-40 are employed, an unlabelled β-amyloid, biotinylated-β-amyloid, and [125I]-labeled β-amyloid. A mixture of the three types of peptides are allowed to aggregate at 37° C. for 4 hrs. At this time, 1 mg of streptavidin coated SPA beads (Amersham) are added to the mixture and allowed to incubate for several hours at 37° C, with measurement of 125I incorporation (into the beads) taken at varying timepoints. To carry out this protocol, large amounts of β1-40 are needed per assay and each assay requires a large amount of time to complete. Thus, this type of assay does not have the high throughput ability needed in the pharmaceutical industry, as well as using potentially hazardous reagents to carry out the protocol.
In the standard Enzyme-Linked Immunosorbant Assay (ELISA) protocol outlined for the detection of α-amyloid aggregation (Howlett et al., FEBS Letters, 417: 249-251. 1997), a polystyrene microtitre plate is coated with a monoclonal antibody to the β-amyloid peptide (e.g. antibody 6E10, Senetek, Napa, Calif.). In a separate microtitre plate, β-amyloid is diluted to a desired concentration in an appropriate buffer and allowed to aggregate overnight in the presence or absence of test compound. After the 24 hr incubation, the aggregation mixture is transferred from the microtitre plate to the p-amyloid antibody coated plate and allowed to bind to antibody. A second, biotinylated 6E10 antibody is then added to the assay plate to bind β-amyloid aggregates. The secondary 6E10 will only be bound if there are β-amyloid molecules present in the assay well bound to other p-amyloid peptides but not to the primary antibody. For detection of the bound biotinylated antibody, Eu3+ labeled streptavidin is added to the wells and detected by excitation at the appropriate wavelength. The amount of Eu3+fluorescence detected will decrease with inhibition of β-amyloid aggregation by the test compound.
While this method is useful in detecting aggregation of β-amyloid peptides, the requirement for peptide-specific monoclonal antibodies limits this assay to availability of the particular antibody and the specificity and binding affinity of the antibody for the peptide.
Berthelier et al, in Anal. Biochem. 295: 227-36. 2001, describe an assay for the detection of polyglutamine aggregate extension where microtiter plate wells are coated with pre-formed polyglutamine aggregates to which are added additional biotinylated-polyglutamine peptides. The rate of incorporation of these newly added peptides is measured using Eu3+labeled-streptavidin to detect bound biotin molecules, corresponding to integrated polyglutamine. This assay, however, does not address whether a test compound affects the prevention of aggregation, or the nucleation event, but rather only provides compounds which modulate continuing polypeptide aggregation.
To this end, Perutz and Windle state in Nature 412: 143-44. 2001, “For any process that occurs on a timescale of years, the controlling step will be nucleation, not growth, and it will occur at random intervals of time.”
Thus, there exists a need in the art for improved materials and methods that address the drawbacks of existing protocols designed to detect polypeptide aggregation, and in doing so expedites development of new therapies and reduces the cost of development.